Reverse-turn mimetics and methods relating thereto

ABSTRACT

Conformationally constrained compounds which mimic the secondary structure of reverse-turn regions of biologically active peptides and proteins are disclosed. Such reverse-turn mimetics have utility in the treatment of cell adhesion-indicated diseases, such as multiple sclerosis, atherosclerosis, asthma and inflammatory bowel disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/344,221 filed Jun. 25, 1999 now U.S. Pat. No. 6,184,223, which is acontinuation-in-part of U.S. application Ser. No. 08/846,432, filed Apr.30, 1997 now U.S. Pat. No. 6,013,458, which is a continuation in part ofU.S. application Ser. No. 08/549,007, filed Oct. 27, 1995 now U.S. Pat.No. 5,929,237.

TECHNICAL FIELD

The present invention relates generally to reverse-turn mimetics,including inhibitors of cell adhesion-mediated disease, as well as to achemical library of reverse-turn mimetics.

BACKGROUND OF THE INVENTION

Cell adhesion is critical to the viability of living organisms. Adhesionholds multicellular tissues together and directs embryonic development.It plays important roles in wound healing, eradication of infection andblood coagulation. Integrins are a family of cell surface proteinsintimately involved in all of these functions. They have been found innearly every type of human cell except red blood cells. Abnormalities inintegrin function contribute to a variety of disorders includinginflammatory diseases, heart attack, stroke, and cancer.

Integrins consist of heterodimers of α and β subunits, non-covalentlybound to each other. These cell surface receptors extend through thecell membrane into the cytoplasm. At least 15 different α and 9different β subunits are known. However, because most α proteinsassociate with only a single β there are about 21 known integrinreceptors. On the cell surface the heads of the two subunits contacteach other to form a binding surface for extracellular protein ligands,allowing attachment to other cells or to the extracellular matrix. Theaffinity of these receptors may be regulated by signals from outside orwithin the cell. For example, recruitment of leukocytes to the site ofinjury or infection involves a series of adhesive interactions. Weakinteraction between endothelial and leukocyte selectins andcarbohydrates mediate transient adhesion and rolling of the leukocytealong the vessel wall. Various chemokines and other trigger factorsreleased by the site of inflammation serve as signals to activateintegrins from a quiescent to a high affinity state. These activatedintegrins then bind their cognate ligands on the surface of theendothelial cells, resulting in strong adhesion and flattening of theleukocyte. Subsequently the leukocyte migrates through the endotheliuminto the tissue below.

Integrin α₄β₁ mediates cell adhesion primarily through binding to eithervascular cell adhesion molecule-1 (VCAM-1) or an alternatively splicedvariant of fibronectin containing the type III connecting segment(IIICS). A variety of cells involved in inflammation express α₄β_(1,)including lymphocytes, monocytes, basophils and eosinophils, but notneutrophils. Monoclonal antibodies to the α₄ subunit have been used tovalidate α₄-containing integrins as potential therapeutic targets inanimal models of rheumatoid arthritis (Barbadillo et al. Springer SeminImmunopathol 16: 427-36, 1995; Issekutz et al. Immunology 88: 569-76,1996), acute colitis (Podolsky et al. J Clin Invest 92: 372-80, 1993),multiple sclerosis (Yednock et al. Nature 356: 63-6, 1992), asthma(Abraham et al. J. Clin. Invest. 93: 776-87, 1994; U.S. Pat. No.5,871,734) and diabetes (Tsukamoto et al. Cell Immunol 165: 193-201,1995). More recently, low molecular weight peptidyl derivatives havebeen produced as competitive inhibitors of α₄β₁ and one has been shownto inhibit allergic airway responses in sheep (Lin et al. J Med Chem 42:920-34, 1999).

It has been shown that a key sequence in IIICS involved in binding toα₄β₁ is the 25 residue peptide CS1, and within that sequence theminimally recognized motif is the tripeptide, LDV. A similar sequence,IDS, has been implicated in the binding of VCAM-1 to α₄β₁. X-ray crystalstructures of an N-terminal two-domain fragment of VCAM-1 show that theIDS sequence is part of an exposed loop linking two beta-strands (Joneset al. Nature 373: 539-44, 1995; Wang et al. Proc Natl Acad Sci U S A92: 5714-8, 1995). Cyclic peptides and derivatives thereof which adoptreverse-turn conformations have proven to be inhibitors of VCAM-1binding to α₄β₁ (WO 96/00581; WO 96/06108; Doyle et al. Int J PeptProtein Res 47: 427-36, 1996). In addition, a number of potent andselective (versus α₅β₁) cyclic peptide-based inhibitors have beendiscovered (Jackson et al. J Med Chem 40: 3359-68, 1997). Severalnon-peptidyl beta-turn mimetics have also been reported to bind α₄β₁with IC₅₀s in the low micromolar range (Souers et al. Bioorg Med ChemLett 8: 2297-302, 1998). Numerous phenylalanine and tyrosine derivativeshave also been disclosed as inhibitors of α₄β₁ (WO 99/06390; WO99/06431; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO99/06437; WO 98/54207; WO 99/10312; WO 99/10313; WO 98/53814; WO98/53817; WO 98/58902) However, no potent and orally available smallmolecule inhibitors have been disclosed.

A related integrin, α₄β₇, is expressed on the surface of lymphocytes andbinds VCAM-1, fibronectin and mucosal addressin cell adhesion molecule 1(MAdCAM-1). Integrin α₄β₇ and MAdCAM mediate recirculation of a subsetof lymphocytes between the blood, gut, and lymphoid tissue. Similar toVCAM-1 and Fibronectin CS-1 there is a tripeptide sequence, LDT, presenton the CD loop of MAdCAM-1 which is important for recognition by α₄β₇.An X-ray crystal structure shows this sequence is also part of a turnstructure (Tan et al. Structure 6: 793-801, 1998). Recent studies haveshown that α₄β₇ may also play a part in diseases such as asthma (Lobb etal. Ann N Y Acad Sci 796: 113-23, 1996), inflammatory bowel disease(Fong et al. Immunol Res 16: 299-311, 1997), and diabetes (Yang et al.Diabetes 46: 1542-7, 1997). In addition, while α₄ integrins appear to bedown-regulated in carcinomas such as cervical and prostate, they appearto be up-regulated in metastatic melanoma (Sanders et al. Cancer Invest16: 329-44, 1998), suggesting that inhibitors of α₄β₁ and α₄β₇ may beuseful as anticancer agents.

Reverse-turns comprise one of three classes of protein secondarystructure and display three (gamma-turn), four (beta-turns), or more(loops) amino acid side chains in a fixed spatial relationship to eachother. Turns have proven important in molecular recognition events (Roseet al. Advances in Protein Chemistry 37: 1-109, 1985) and haveengendered a burgeoning field of research into small molecule mimeticsof them (e.g. Hanessian et al. Tetrahedron 53: 12789-12854, 1997). Manymimetics have either been external turn-mimetics which do not allow forthe display of all the physiologically relevant side-chains (e.g.Freidinger et al. Science 210: 656-8, 1980) or small, conformationallymobile cyclic peptide derivatives (e.g. Viles et al. Eur J Biochem 242:352-62, 1996). However, non-peptide compounds have been developed whichclosely mimic the secondary structure of reverse-turns found inbiologically active proteins or peptides. For example, U.S. Pat. Nos.5,475,085, 5,670,155 and 5,672,681, to Kahn and published PCT WO94/03494to Kahn all disclose conformationally constrained, non-peptidiccompounds which mimic the three-dimensional structure of reverse-turns.More recently, published PCT WO97/15577 to Kahn and PCT WO98/49168 toKahn et al. have disclosed additional, highly constrained bicyclicheterocycles as reverse-turn mimetics. Nevertheless, as no one templatecan mimic every type of turn, there remains a need in the art foradditional reverse-turn templates and methods for their use.

While significant advances have been made in the synthesis andidentification of conformationally constrained, reverse-turn mimetics,there is still a need in the art for small molecules that mimic thesecondary structure of peptides. In addition, there is a need in the artfor techniques for synthesizing libraries of such mimetics and screeningthe library members against biological targets to identify bloactivelibrary members. Further, there is a need in the art for small, orallyavailable inhibitors of integrins, for use in treating inflammatorydiseases and cardiovascular diseases, as well as some cancers. Inparticular there is a need for inhibitors of α₄β₁ and α₄β₇, for use inthe treatment of rheumatoid arthritis, asthma, diabetes and inflammatorybowel disease. The present invention fulfills these needs, and providesfurther related advantages.

SUMMARY OF THE INVENTION

In brief, the present invention is directed to conformationallyconstrained compounds which mimic the secondary structure ofreverse-turn regions of biologically active peptides and proteins. Thisinvention also discloses libraries containing such compounds, as well asthe synthesis and screening thereof. Furthermore, the inventiondiscloses the use of reverse-turn mimetics for the treatment of celladhesion-mediated diseases.

The compounds of the present invention have the following generalstructure (I):

wherein Y is selected from —CH(R₅)-A-N(R₁)—, -A-N(R₁)—CH(R′)—,-A-N(R₁)—C(═O)—, -A-C(═O)—N(R₁)—, -A-CH(R₁)—O—, and -A-CH(R₁)—N(R′)—; Ais —(CHR′)_(n)—; B is —(CHR″)_(m)—; n=0, 1 or 2; m=1, 2 or 3; and anytwo adjacent CH groups or adjacent NH and CH groups on the bicyclic ringmay optionally form a double bond; and wherein R′, R″, R₁, R₂, R₃, R₄and R₅ are as defined in the following detailed description.

In the embodiment wherein Y is —CH(R₅)-A-N(R₁)—, the compounds of thisinvention have the following structure (I′):

wherein A and B are as defined above, and R₁, R₂, R₃, R₄ and R₅ are asdefined in the following detailed description.

In the embodiment wherein Y is -A-N(R₁)—CH(R′)—, the compounds of thisinvention have the following structure (I″):

wherein A and B are as defined above, and R′, R₁, R₂, R₃ and R₄ are asdefined in the following detailed description.

In the embodiment wherein Y is -A-N(R₁)—C(═O)—, the compounds of thisinvention have the following structure (I″′):

wherein A and B are as defined above, and R₁, R₂, R₃ and R₄ are asdefined in the following detailed description.

In the embodiment wherein Y is -A-C(═O)—N(R₁)—, the compounds of thisinvention have the following structure (I″″):

wherein A and B are as defined above, and R₁, R₂, R₃ and R₄ are asdefined in the following detailed description.

In the embodiment wherein Y is -A-CH(R₁)—O—, the compounds of thisinvention have the following structure (I″″′):

wherein A and B are as defined above, and R₁, R₂, R₃ and R₄ are asdefined in the following detailed description.

In the embodiment wherein Y is -A-CH(R₁)—N(R′)—, the compounds of thisinvention have the following structure (I″″″):

wherein A and B are as defined above, and R′, R₁, R₂, R₃ and R₄ are asdefined in the following detailed description.

The present invention is also directed to libraries containing compoundsof structure (I) above, as well as methods for synthesizing suchlibraries and methods for screening the same to identify biologicallyactive compounds. Methods of use for treating cell-adhesion-mediateddisease with the compounds of this invention are described. Compositionscontaining a compound of this invention in combination with apharmaceutically acceptable carrier or diluent are also disclosed.

These and other aspects of this invention will be apparent uponreference to the attached figures and following detailed description. Tothis end, various references are set forth herein which describe in moredetail certain procedures, compounds and/or compositions, and areincorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the percent inhibition of radioligand binding to δand μ opiate receptors of a representative reverse-turn mimetic of thisinvention as a function of concentration.

FIGS. 2-9 illustrate representative reaction schemes for the synthesisof reverse-turn mimetics of this invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to reverse-turn mimetics and chemicallibraries containing reverse-turn mimetics. The reverse-turn mimetics ofthe present invention are useful as bioactive agents, including (but notlimited to) use as diagnostic, prophylactic and/or therapeutic agents.The reverse-turn mimetic libraries of this invention are useful in theidentification of such bioactive agents. In the practice of the presentinvention, the libraries may contain from tens to hundreds to thousands(or greater) of individual reverse-turn mimetics (also referred toherein as “members”).

In one aspect of the present invention, a reverse-turn mimetic isdisclosed having the following structure (I):

wherein Y is selected from —CH(R₅)-A-N(R₁)—, -A-N(R₁)—CH(R′)—,-A-N(R₁)—C(═O)—, -A-C(═O)—N(R₁)—, -A-CH(R₁)—O— and -A-CH(R₁)—N(R′)—; Ais —(CHR′)—; B is —(CHR″)_(m)—; n=0, 1 or 2; m=1, 2 or 3; and any twoadjacent CH groups or adjacent NH and CH groups on the bicyclic ring mayoptionally form a double bond; and wherein R′, R″, R₁, R₂, R₃, R₄ and R₅are as defined below.

In structures (I′) through (I″″″) above a solid line designation forattachment of the various R groups to a carbon atom on the fusedbicyclic ring indicates that these R groups may lie either above orbelow the plane of the page. If a reverse-turn mimetic of this inventionis intended to mimic a reverse-turn of naturally occurring amino acids(i.e., “L-amino acids”), the R groups would generally lie below theplane of the page (i.e., “ R”) in Structure (I). However, if thereverse-turn mimetic of this invention is intended to mimic areverse-turn containing one or more D-amino acids, then thecorresponding R group or groups would lie above the plane of the page(i.e., “ R”) in Structure (I).

In one embodiment, R₁ and R₄ are the same or different and represent theremainder of the compound, and R′, R″, R₂, R₃, and R₅ are the same ordifferent and independently selected from an amino acid side chainmoiety or derivative thereof. With regard to R′ and R″, it should beunderstood that each occurrence of R′ and R″ is independently selectedfrom amino acid side chain moieties or derivatives thereof. For example,when m=2, B is a —CHR″CHR″— moiety. In this instance. both occurrencesof R″ are independently selected, and may be the same or different.Thus, if the first occurrence of R″ is hydrogen and the second methyl, Bwould have the structure —CH₂CH(CH₃)—.

As used herein, the term “remainder of the compound” means any moiety,agent, compound, support, molecule, linker, amino acid, peptide orprotein covalently attached to the reverse-turn mimetic at either the R₁and/or R₄ positions. This term also includes amino acid side chainmoieties and derivatives thereof.

As used herein, the term “amino acid side chain moiety” represents anyamino acid side chain moiety present in naturally occurring proteinsincluding (but not limited to) the naturally occurring amino acid sidechain moieties identified in Table 1. Other naturally occurring aminoacid side chain moieties of this invention include (but are not limitedto) the side chain moieties of 3,5-dibromotyrosine, 3,5-diiodotyrosine,hydroxylysine, γ-carboxyglutamate, phosphotyrosine and phosphoserine. Inaddition, glycosylated amino acid side chains may also be used in thepractice of this invention, including (but not limited to) glycosylatedthreonine, serine and asparagine.

TABLE 1 Amino Acid Side Chain Moieties Amino Acid Side Chain MoietyAmino Acid —H Glycine —CH₃ Alanine —CH(CH₃)₂ Valine —CH₂CH(CH₃)₂ Leucine—CH(CH₃)CH₂CH₃ Isoleucine —(CH₂)₄NH₃ ⁺ Lysine —(CH₂)₃NHC(NH₂)NH₂ ⁺Arginine

Histidine —CH₂COO⁻ Aspartic acid —CH₂CH₂COO⁻ Glutamic acid —CH₂CONH₂Asparagine —CH₂CH₂CONH₂ Glutamine

Phenylalanine

Tyrosine

Tryptophan —CH₂SH Cysteine —CH₂CH₂SCH₃ Methionine —CH₂OH Serine—CH(OH)CH₃ Threonine

Proline

Hydroxyproline

In addition to naturally occurring amino acid side chain moieties, theamino acid side chain moieties of the present invention also includevarious derivatives thereof. As used herein, a “derivative” of an aminoacid side chain moiety includes modifications and/or variations tonaturally occurring amino acid side chain moieties. For example, theamino acid side chain moieties of alanine, valine, leucine, isoleucineand phenylalanine may generally be classified as lower chain alkyl,aryl, or aralkyl moieties. Derivatives of amino acid side chain moietiesinclude other straight chain or branched, cyclic or noncyclic,substituted or unsubstituted, saturated or unsaturated lower chainalkyl, aryl or aralkyl moieties.

As used herein, “lower chain alkyl moieties” contain from 1-12 carbonatoms, “lower chain aryl moieties” contain from 6-12 carbon atoms and“lower chain aralkyl moieties” contain from 7-12 carbon atoms. Thus, inone embodiment, the amino acid side chain derivative is selected from aC₁₋₁₂ alkyl, a C₆₋₁₂ aryl and a C₇₋₁₂ aralkyl, and in a more preferredembodiment, from a C₁₋₇ alkyl, a C₆₋₁₀ aryl and a C₇₋₁₁ aralkyl.

Amino side chain derivatives of this invention further includesubstituted derivatives of lower chain alkyl, aryl, and aralkylmoieties, wherein the substituent is selected from (but are not limitedto) one or more of the following chemical moieties: —OH, —OR, —COOH,—COOR, —CONH₂, —NH₂, —NHR, —NRR, —SH, —SR, —SO₂R, —SO₂H, —SOR andhalogen (including F, Cl, Br and I), wherein each occurrence of R isindependently selected from straight chain or branched, cyclic ornoncyclic, substituted or unsubstituted, saturated or unsaturated lowerchain alkyl, aryl and aralkyl moieties. Moreover, cyclic lower chainalkyl, aryl and aralkyl moieties of this invention include naphthalene,as well as heterocyclic compounds such as thiophene, pyrrole, furan,imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine,pyridine, pyrimidine, purine, quinoline, isoquinoline and carbazole.Amino acid side chain derivatives further include heteroalkylderivatives of the alkyl portion of the lower chain alkyl and aralkylmoieties, including (but not limited to) alkyl and aralkyl phosphonatesand silanes.

Representative R₁ and R₄ moieties specifically include (but are notlimited to) —OH, —OR, —COR, —COOR, —CON₂, —CONR, —CONRR, —NH₂, —NHR,—NRR, —SO₂R and —COSR, wherein each occurrence of R is as defined above.

In a further embodiment, and in addition to being an amino acid sidechain moiety or derivative thereof (or the remainder of the compound inthe case of R₁ and R₄), R₁, R₂, R₃, R₄, or R₅ may be a linkerfacilitating the linkage of the compound to another moiety or compound.For example, the compounds of this invention may be linked to one ormore known compounds, such as biotin, for use in diagnostic or screeningassay. Furthermore, R₁, R₂, R₃, R₄ or R₅ may be a linker joining thecompound to a solid support (such as a support used in solid phasepeptide synthesis) or alternatively, may be the support itself. In thisembodiment, linkage to another moiety or compound, or to a solidsupport, is preferable at the R₁ or R₄ position, and more preferably atthe R₄ position.

In the embodiment where Y is —CH(R₅)-A-N(R₁)—, the reverse-turn mimetichas the following structure (I′):

wherein A, B, R₁, R₂, R₃, R₄ and R₅ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂,R₃ and R₅ are individually selected from an amino acid side chainmoiety.

In a more specific embodiment of structure (I′), A is —(CH₂)_(n)—, B is—(CH₂)_(m)—, and the reverse-turn mimetic has the following structure(Ia′):

wherein n, m, R₁, R₂, R₃, R₄ and R₅ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂,R₃ and R₅ are individually selected from an amino acid side chainmoiety.

In a yet more specific embodiment of structure (I′), A is —(CH₂)_(n)—, Bis —(CH₂)_(m)—, n is 0, m is 1 and the reverse-turn mimetic has thefollowing structure (Ib′):

wherein R₁, R₂, R₃, R₄ and R₅ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂,R₃ and R₅ are individually selected from an amino acid side chainmoiety. In another preferred embodiment, R₁ is selected from ROC(O)—,RSO₂— and RNHC(O)—, wherein R is as defined above. In a more preferredembodiment, R₁ is selected from ROC(O)—, RSO₂— and RNHC(O)— and R isselected from substituted or unsubstituted lower chain aryl and lowerchain aralkyl moieties. In another specific embodiment, R₂ and R₅ areindependently selected from lower chain alkyl moieties, substituted withCOOH or COOR, wherein R is as defined above. In another specificembodiment, R₃ is selected from substituted or unsubstituted lower chainaryl and lower chain aralkyl moieties.

In the embodiment where Y is -A-N(R₁)—CH(R′)—, the reverse-turn mimetichas the following structure (I″).

wherein A, B, R₁, R₂, R₃, R₄ and R′ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂,R₃ and R′ are individually selected from an amino acid side chainmoiety.

In an embodiment of structure (I″) where two adjacent CH groups on thebicyclic ring form a double bond, the reverse-turn mimetics of thisinvention include the following structure (Ia″):

wherein A, B, R₁, R₂, R₃, R₄ and R′ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, R₂ and R₃are independently selected from an amino acid side chain moiety, and R′is hydrogen.

In a more specific embodiment of structure (Ia″), A is —(CH₂)_(n)—, B is—(CH₂)_(m)—, R′ is hydrogen, and the reverse-turn mimetic has thefollowing structure (Ib″):

wherein n, m, R₁, R₂, R₃ and R₄ are as defined above.

In the embodiment where Y is -A-N(R₁)—C(═O)—, the reverse turn mimetichas the following structure (I″′):

wherehin A, B, R₁, R₂, R₃ and R₄ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂and R₃ are independently selected from an amino acid side chain moiety.

In a more specific embodiment of structure (I″′), A is —(CH₂)_(n)—, B is—(CH₂)_(m)—, and the reverse-turn mimetic has the following structure(Ia″′):

wherein n, m, R₁, R₂, R₃ and R₄ are as defined above.

In the embodiment where Y is -A-C(═O)—N(R₁)—, the reverse turn mimetichas the following structure (I″″):

wherein R₁, R₂, R₃ and R₄ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂and R₃ are independently selected from an amino acid side chain moiety.

In a more specific embodiment of structure (I″″), A is —(CH₂)_(n)—, B is—(CH₂)_(m)—, and the reverse-turn mimetic has the following structure(Ia″″):

wherein n, m, R₁, R₂, R₃ and R₄ are as defined above.

In the embodiment where Y is -A-CH(R₁)—O—, the reverse-turn mimetic hasthe following structure (I″″′):

wherein R₁, R₂, R₃ and R₄ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂and R₃ are independently selected from an amino acid side chain moiety.

In a more specific embodiment of structure (I″″′), A is —(CH₂)_(n)—, Bis —(CH₂)_(m)—, and the reverse-turn mimetic has the following structure(Ia″″′):

wherein n, m, R₁, R₂, R₃ and R₄ are as defined above.

In the embodiment where Y is -A-CH(R₁)—N(R′)—, and adjacent NH and CHgroups on the bicyclic ring form a double bond, the reverse-turnmimetics of this invention include the following structure (Ia″″″):

wherein A, B, R₁, R₂, R₃ and R₄ are as defined above. In a preferredembodiment, R₁ and R₄ represent the remainder of the compound, and R₂and R₃ are independently selected from an amino acid side chain moiety.

In a more specific embodiment of structure (Ia″″″), A is —(CH₂)_(n)—, Bis —(CH₂)_(m)—, and the reverse-turn mimetic has the following structure(Ib″″″):

wherein n, m, R₁, R₂, R₃ and R₄ are as defined above.

In preferred embodiment of structure (I), R₁ is selected from ROC(o)-,RSO₂— AND RHNC(O)—, wherein R is as defined above. In a more specificembodiment, R₁ is selected from ROC(O)—, RSO₂— and RHNC(O) and R isselected from substituted or unsubstituted lower chain aryl and lowerchain aralykl moieties.

In a specific embodiment of structure (I), R₂ and R₅ are independentlyselected from lower chain alkyl moieties, substituted with COOH or COOR,wherein R is as defined above. In another specific embodiment ofstructure (I), R₂ and R₅ are independently selected from H— and RC(O)NH—wherein R is as defined above.

In another specific embodiment of structure (I), R₃ is selected fromsubstituted or unsubstituted lower chain aryl and lower chain aralkylmoieties.

In another specific embodiment of structure (I), R₃ is selected fromsubstituted or unsubstituted lower chain aryl and lower chain aralkyl,including hererocyclic, moieties.

The reverse-turn mimetics of the present invention may be prepared byutilizing appropriate starting component molecules (hereinafter referredto as “component pieces”). Briefly, in the synthesis of reverse turnmimetics having structure (I′), first and second component pieces arecoupled to form a combined first-second intermediate, third and fourthcomponent pieces are coupled to form a combined third-fourthintermediate (or, if commercially available, a single third intermediatemay be used), the combined first-second intermediate and third-fourthintermediate (or third intermediate) are then coupled to provide afirst-second-third-fourth intermediate (or first-second-thirdintermediate) which is cyclized to yield the reverse-turn mimetics ofthis invention. Alternatively, the reverse-turn mimetics of structure(I′) may be prepared by sequential coupling of the individual componentpieces either stepwise in solution or by solid phase synthesis ascommonly practiced in solid phase peptide synthesis.

Within the context of the present invention, a “first component piece”has the following structure 1:

where R₄ and B are as defined above, and R is a protective groupsuitable for use in peptide synthesis. Suitable R groups include alkylgroups and, in a preferred embodiment, R is a methyl group. Such firstcomponent pieces may be readily synthesized by reductive amination bymating CH(OR)₂—(CH₂)m-CHO with H₂N—R₄, or by displacement fromCH(OR)₂—(CH₂)m-Br. Alternatively, one of the R-groups may be a linkerand resin. Polystyrene resins, such as those typically used in peptidesynthesis and containing the Wang linker(4-hydroxymethylphenoxybutyrate), are suitable.

A “second component piece” of this invention has the following structure2:

where R₃ is as defined above, P is an amino protective group suitablefor use in peptide synthesis, and X represents the leaving group of theactivated carboxylic acid group. Preferred protective groups includet-butyl dimethylsilyl (TBDMS), BOC, FMOC, and Alloc (allyloxycarbonyl).N-Protected amino acids are commercially available. For example, FMOCamino acids are available from a variety of sources. The conversion ofthese compounds to the second component pieces of this invention may bereadily achieved by activation of the carboxylic acid group of theN-protected amino acid. Suitable activated carboxylic acid groupsinclude acid halides where X is a halide such as chloride or bromide,acid anhydrides where X is an acyl group such as acetyl, reactive esterssuch as an N-hydroxybenzotriazole esters, N-hydroxysuccinimide estersand pentafluorophenyl esters, and other activated intermediates such asthe active intermediate formed in a coupling reaction using acarbodiimide such as dicyclohexylcarbodiimide (DCC) ordiisopropylcarbodiimide (DIC).

In the case of the azido derivative of an amino acid serving as thesecond component piece, such compounds may be prepared from thecorresponding amino acid by the reaction disclosed by Zaloom et al. (J.Org. Chem. 46:5173-76, 1981).

A “third component piece” of this invention has the following structure3:

where R₂ and R₅ are as defined above, and P is a carboxylic acidprotective group such as a methyl or t-butyl group.

A “fourth component piece” of this invention has the following structure4:

R₁—NH₂   4

where R₁ is as defined above. Suitable fourth component pieces arecommercially available from a variety of sources. Alternatively, thefourth component pieces may be readily prepared by standard organicsynthetic techniques commonly utilized for the synthesis of primaryamines.

More specifically, the reverse-turn mimetics of this invention ofstructure (I′) are synthesized by reacting a first component piece witha second component piece to yield a combined first-second intermediate,followed by either reacting the combined first-second intermediate withthird and fourth component pieces sequentially, or reacting theintermediate with a combined third-fourth intermediate to provide acombined first-second-third-fourth intermediate, and then cyclizing thisintermediate to yield the reverse-turn mimetic.

The general synthesis of a reverse-turn mimetic having structure I′ maybe synthesized by the following technique. A first component piece 1 iscoupled to a second component piece 2 to yield, after N-deprotection, acombined first-second intermediate 1-2 as illustrated below:

The synthesis of the reverse-turn mimetic may be convergent, in whichcase a combined third-fourth intermediate 3-4 is prepared from thecoupling of a third component piece 3 with a fourth component piece 4 toyield, after O-deprotection, a combined third-fourth intermediate 3-4 asillustrated below:

In the case where n of structure (I) above is 1 or 2, an intermediate ofthe following structure 3-4′ can be made as follows:

wherein A is —(CHR′)_(n)—. Intermediate 3-4′ may then be employed inplace of intermediate 3-4 in the following reactions to yield areverse-turn mimetic of this invention having structure (I′).Alternatively, in the case where n of structure (I) above is 1, 2 or 3,3-4′ may be made from a beta-, gamma- or delta-amino acid derivativewhich is acylated or sulfonylated and then O-deprotected as follows:

Coupling of the combined intermediates 1-2 and 3-4 provides intermediate1-2-3-4 which, upon cyclization, yield the reverse-turn mimetic (I′) asillustrated below:

The syntheses of representative component pieces of this invention aredescribed in Example 1. The syntheses of representative combinedfirst-second and third-fourth intermediates are described in Examples 2and 3, respectively. The coupling of these intermediates to form arepresentative combined first-second-third-fourth intermediate isdescribed in Example 4. The cyclization of this intermediate to form arepresentative reverse-turn mimetic is described in Example 5.

In a preferred embodiment, the reverse-turn mimetic of structure (Ia′)may be made in solution according to the reaction scheme set forth inFIG. 2. In another preferred embodiment, the reverse-turn mimetic ofstructure (Ia′) may be made on solid-phase according to the reactionscheme set forth in FIG. 8 and described in Example 8. In a morepreferred embodiment, the reverse-turn mimetic of structure (Ia′) may bemade on solid-phase according to the reaction scheme set forth in FIG. 9and described in Example 10.

The reverse-turn mimetics of structures (I″) through (I″″″) may be madeby techniques analogous to the modular component synthesis disclosedabove, but with appropriate modifications to the component pieces. Morespecifically, the reverse-turm mimetics of structures (I″) through(I″″″) may be made by the reaction schemes set forth in FIGS. 3-7. Inparticular, the reverse-turn mimetics of structures (Ib″), (Ia″′),(Ia″″), (Ia″″′) and (Ib″″″) may be made by the representative reactionschemes set forth in FIGS. 3, 4, 5, 6 and 7, respectively.

In another aspect of this invention, libraries containing reverse-turnmimetics of the present invention are disclosed. Once assembled, thelibraries of the present invention may be screened to identifyindividual members having bioactivity. Such screening of the librariesfor bioactive members may involve, for example, evaluating the bindingactivity of the members of the library or evaluating the effect thelibrary members have on a functional assay. Screening is normallyaccomplished by contacting the library members (or a subset of librarymembers) with a target of interest, such as, for example, an antibody,enzyme, receptor or cell line. Library members which are capable ofinteracting with the target of interest are referred to herein as“bioactive library members” or “bioactive mimetics”. For example, abioactive mimetic may be a library member which is capable of binding toan antibody or receptor, which is capable of inhibiting an enzyme, orwhich is capable of eliciting or antagonizing a functional responseassociated, for example, with a cell line. In other words, the screeningof the libraries of the present invention determines which librarymembers are capable of interacting with one or more biological targetsof interest. Furthermore, when interaction does occur, the bioactivemimetic (or mimetics) may then be identified from the library members.The identification of a single (or limited number) of bioactivemimetic(s) from the library yields reverse-turn mimetics which arethemselves biologically active, and thus useful as diagnostic,prophylactic or therapeutic agents, and may further be used tosignificantly advance identification of lead compounds in these fields.

Synthesis of the peptide mimetics of the library of the presentinvention may be accomplished using known peptide synthesis techniques,in combination with the first, second and third component pieces of thisinvention.) More specifically, any amino acid sequence may be added asany of the R₁, R₂, R₃, R₄ or R₅ moieties of the conformationallyconstrained reverse-turn mimetic. Preferably the amino acid sequence maybe added as the R₁ or R₄ moieties. To this end, the mimetics may besynthesized on a solid support (such as polystyrene utilizing4-hydroxymethylphenoxybutyrate as a linker) by known techniques (see,e.g., John M. Stewart and Janis D. Young, Solid Phase Peptide Synthesis,1984, Pierce Chemical Comp., Rockford, Ill.; Atherton, E., Shepard, R.C. Solid Phase Pepetide Synthesis: A Practical Approach; IRL: Oxford,1989) or on a silyl-linked resin by alcohol attachment (see Randolph etal., J. Am Chem. Soc. 117:5712-14, 1995).

In addition, a combination of both solution and solid phase synthesistechniques may be utilized to synthesize the peptide mimetics of thisinvention. For example, a solid support may be utilized to synthesizethe linear peptide sequence up to the point that the conformationallyconstrained reverse-turn is added to the sequence. A suitableconformationally constrained reverse-turn mimetic which has beenpreviously synthesized by solution synthesis techniques may then beadded as the next “amino acid” to the solid phase synthesis (i.e., theconformationally constrained reverse-turn mimetic, which has at leasttwo reactive sites, may be utilized as the next residue to be added tothe linear peptide). Upon incorporation of the conformationallyconstrained reverse-turn mimetic into the sequence, additional aminoacids may then be added to complete the peptide bound to the solidsupport. Alternatively, the linear N-terminus and C-terminus protectedpeptide sequences may be synthesized on a solid support, removed fromthe support, and then coupled to the conformationally constrainedreverse-turn mimetic in solution using known solution couplingtechniques.

In another aspect of this invention, methods for constructing thelibraries are disclosed. Traditional combinatorial chemistry andparallel synthesis techniques (see, e.g., The Combinatorial Index Bunin,Academic Press, New York, 1998; Gallop et al., J. Med. Chem.37:1233-1251, 1994) permit a vast number of compounds to be rapidlyprepared by the sequential combination of reagents to a basic molecularscaffold. For example, the above disclosed synthesis may be carried outusing the directed sorting technique of Nicolaou and coworkers(Nicolaou, Xiao et al. Angew. Chem. Int. Ed. 34: 2289-2291, 1995).Presently, equipment for this technique is commercially available fromIRORI (La Jolla, Calif.). Alternatively, the above disclosed synthesismay be carried out by parallel synthesis using a 48- or 98-well plateformat wherein each well contains a fritted outlet for draining solventsand reagents (A Practical Guiide to Combinatorial Chemistry Czarnik andDeWitt, Eds., American Chemical Society, Washington, D.C., 1997).Robbins (Sunnyvale, Calif.), Charybdis (Carlsbad, Calif.) and Bohdan(Chicago, Ill.) presently offer suitable equipment for this technique.

In a further aspect of this invention, methods for screening thelibraries for bioactivity and isolating bioactive library members aredisclosed. The libraries of the present invention may be screened forbioactivity by a variety of techniques and methods. Generally, thescreening assay may be performed by (1) contacting a library with abiological target of interest, such as a receptor, and allowing bindingto occur between the mimetics of the library and the target, and (2)detecting the binding event by an appropriate assay, such as by thecolorimetric assay disclosed by Lam et al. (Nature 354:82-84, 1991) orGriminski et al. (Biotechnology 12:1008-1011, 1994). In a preferredembodiment, the library members are in solution and the target isimmobilized on a solid phase. Alternatively, the library may beimmobilized on a solid phase and may be probed by contacting it with thetarget in solution.

As mentioned above, the reverse-turn mimetics of the present inventionare useful as bioactive agents, such as diagnostic, prophylactic, andtherapeutic agents. The opiate receptor binding activity ofrepresentative reverse-turn mimetics is presented in Example 9. In thisexample, the reverse-turn mimetics of this invention were found toeffectively inhibit the binding of a radiolabeled enkephalin derivativeto the δ and μ opiate receptors. The data demonstrates the utility ofthese reverse-turn mimetics as receptor antagonists and as potentialanalgesic agents. In a further embodiment, the integrin binding activityof representative reverse-turn mimetics is presented in Example 11. Inthis example, the reverse-turn mimetics were found to effectivelydisplace CS1 peptide from Ramos cells. The data thus indicate theability of reverse turn mimetics to antagonize α₄β₁ integrins and serveas potential anti-inflammatory agents.

In another aspect, the present invention encompasses pharmaceuticalcompositions prepared for storage or administration which comprise atherapeutically effective amount of a compound of the present inventionin a pharmaceutically acceptable carrier or diluent. Therapy withinhibitors of cell adhesion is indicated for the treatment andprevention of a variety of inflammatory conditions, particularlyrheumatoid arthritis, inflammatory bowel disease and asthma. Thoseexperienced in this field are readily aware of the circumstancesrequiring anti-inflammatory therapy. In addition, therapy withinhibitors of cell adhesion are indicated for any condition in which anexcess of integrin-mediated cell adhesion is a contributing factor, suchas, for example, atherosclerosis.

The “therapeutically effective amount” of a compound of the presentinvention will depend on the route of administration, the type ofwarm-blooded animal being treated, and the physical characteristics ofthe specific animal under consideration. These factors and theirrelationship to determining this amount are well known to skilledpractitioners in the medical arts. This amount and the method ofadministration can be tailored to achieve optimal efficacy but willdepend on such factors as weight, diet, concurrent medication and otherfactors which as noted those skilled in the medical arts will recognize.

The “therapeutically effective amount” of the compound of the presentinvention can range broadly depending upon the desired affects and thetherapeutic indication. Typically, dosages will be between about 0.01mg/kg and 100 mg/kg body weight, preferably between about 0.01 and 10mg/kg, body weight.

“Pharmaceutically acceptable carriers” for therapeutic use, includingdiluents, are well known in the pharmaceutical art, and are described,for example, in Remingtons Pharmaceiutical Sciences, Mack Publishing Co.(Gennaro Ed. 1985). For example, sterile saline and phosphate-bufferedsaline at physiological pH may be used. Preservatives, stabilizers, dyesand even flavoring agents may be provided in the pharmaceuticalcomposition. For example, sodium benzoate, sorbic acid and esters ofp-hydroxybenzoic acid may be added as preservatives. In addition,antioxidants and suspending agents may be used.

Compounds of the present invention are useful for prevention andtreatment of any condition in which an excess of integrin-mediated celladhesion is a contributing factor. In particular, the compounds of thepresent invention are useful as agents for the prevention and treatmentof inflammation and related conditions. In the practice of the methodsof this invention, a composition containing a therapeutically effectiveamount of a compound of this invention is administered to a warm-bloodedanimal in need thereof. For example, the compounds of this invention maybe administered to a warm-blooded animal that has been diagnosed with,or is at risk of developing a condition selected from rheumatoidarthritis, atherosclerosis, Alzheimer's disease, AIDS dementia, ARDS,asthma, allergies, inflammatory bowel disease, CNS inflammation, atopicdermatitis, type I diabetes, encephalitis, myocardial ischemia, multiplesclerosis, meningitis, nephritis, restenosis, retinitis, psoriasis,stroke and tumor metastasis.

Multiple sclerosis (MS) is a progressively debilitating autoimmunedisease of the central nervous system. Presently the exact antigentriggering the immune response is unknown. However, macrophages appearto attack and initiate the destruction of the fatty myelin sheathssurrounding nerve fibers in the brain. In an animal model of MS(experimental allergic encephalomyelitis) murine monoclonal antibodiesto α₄β₁ blocked adhesion of the leukocytes to the endothelium, andprevented inflammation of the central nervous system and subsequentparalysis of the animals (Yednock, Cannon et al. Nature 356: 63-6,1992).

The compounds of the present invention may be used singularly, as acombination of two or more compounds, or in combination with other knowninhibitors of inflammation. For example the compounds of this inventionmay be used therapeutically with corticosteroids, non-steroidalanti-inflammatory agents, COX-2 inhibitors, matrix metalloproteaseinhibitors or lipoxygenase inhibitors. The compounds of the inventioncan be administered in such oral forms as tablets, capsules (each ofwhich includes sustained release or timed release formulations), pills,powders, granules, elixers, tinctures, suspensions, syrups, andemulsions. Likewise, they may be administered in intravenous (bolus orinfusion), intraperitoneal, subcutaneous, intranasal, intrarectal orintramuscular form, all using forms well known to those of ordinaryskill in the pharmaceutical arts. The compounds may be administeredintraocularly or topically as well as orally or parenterally.

The compounds of this invention may be administered by inhalation, andthus may be delivered in the form of an aerosol spray from pressurizedpacks or nebulizers. The compounds may also be delivered as powderswhich may be formulated and the powder composition may be inhaled withthe aid of an insufflation powder inhaler device. A preferred deliverysystem for inhalation is the metered dose inhalation aerosol, which maybe formulated as a suspension or solution of a compound of the inventionin suitable propellants, such as fluorocarbons or hydrocarbons. Anotherpreferred delivery system is the dry powder inhalation aerosol, whichmay be formulated as a dry powder of a compound of this invention withor without additional excipients.

The compounds of the invention can be administered in the form of adepot injection or implant preparation which may be formulated in such amanner as to permit a sustained release of the active ingredient. Theactive ingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers or synthetic silicones, for example, Silastic, silicone rubberor other polymers manufactured by the Dow-Corning Corporation.

The compounds of the invention can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The compounds of this invention may also be delivered by the use ofmonoclonal antibodies as individual carriers to which the compoundmolecules are coupled. The integrin inhibitors may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can includepolyvinlypyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartarnide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the integrininhibitors may be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example, polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydihydropyrans, polycyanoacrylates and cross linked oramphipathic block copolymers of hydrogels.

The dose and method of administration can be tailored to achieve optimalefficacy but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize. When administration is to be parenteral, such asintravenous on a daily basis, injectable pharmaceutical compositions canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions.

Tablets suitable for oral administration of active compounds of theinvention can be prepared as follows:

Amount-mg Active Compound 25.0 50.0 100.0 Microcrystalline cellulose37.25 100.0 200.0 Modified food corn starch 37.25 4.25 8.5 Magnesiumstearate 0.50 0.75 1.5

All of the active compound, cellulose, and a portion of the corn starchare mixed and granulated to 10% corn starch paste. The resultinggranulation is sieved, dried and blended with the remainder of the cornstarch and the magnesium stearate. The resulting granulation is thencompressed into tablets containing 25.0, 50.0, and 100.0 mg,respectively, of active ingredient per tablet.

An intravenous dosage form of the above-indicated active compounds maybe prepared as follows:

Active Compound 0.5-10.0 mg Sodium Citrate 5-50 mg Citric Acid 1-15 mgSodium Chloride 1-8 mg  Water for Injection (USP) q.s. to 1 mL

Utilizing the above quantities, the active compound is dissolved at roomtemperature in a previously prepared solution of sodium chloride, citricacid, and sodium citrate in Water for Injection (USP, see page 1636 ofUnited States Pharmacopoeia/National Formulary for 1995, published byUnited States Pharmacopoeia Convention, Inc., Rockville, Md., copyright1994).

The following examples are provided for purposes of illustration, notlimitation.

EXAMPLES Example 1 Synthesis of Component Pieces

In this example, the synthesis of representative component pieces whichmay be combined to form the reverse-turn mimetics of the presentinvention is disclosed.

A. Representative First Component Pieces

A first component piece having the following structure 1 was utilized:

where R₄ is as defined above, and R represents a protective groupsuitable for use in peptide synthesis. Suitable R groups include alkylgroups and, in a preferred embodiment, R is a methyl group.

Generally, the first component piece is prepared by N-alkylation of anamine with a dialkylacetal of a 2-haloethanal. The synthesis of arepresentative first component piece from phenethylamine and thedimethylacetal of 2-bromoethanal is depicted schematically below.

In the procedure, 24 ml (3.43 ml, 20.3 mmol) of bromide and 2.8 ml (2.71g. 22.3 mmol) phenethylamine was added 40 ml freshly distilled THF in a150 ml argon charged round-bottom flask equipped with a refluxcondenser. The reaction was heated at a gentle reflux for 24 hours, thenvolatiles were removed under reduced pressure and the residue wasdissolved in 200 ml dichloromethane. The organic layer was washed with2×100 ml sat. aq. sodium bicarbonate, sat. aq. sodium chloride, anddried over anhydrous sodium sulfate. Volatiles were removed underreduced pressure and the residue dried for 3 hrs. under high vacuum toyield 3.5 g (83%) first component piece 1a (m=1) as a light brown oilused without further purification.

B. Representative Second Component Pieces

A representative second component piece of this invention is a reactiveN-protected amino acid having an activated carboxylic acid group, or anazido derivative of an amino acid, as represented by the followingstructure 2:

where R₃ is as defined above, P is an amino protective group suitablefor use in peptide synthesis, and X represents the leaving group of theactivated carboxylic acid group. Preferred protective groups includet-butyl dimethylsilyl (TBDMS), BOC, FMOC, and Alloc (allyloxycarbonyl).N-Protected amino acids are commercially available. For example, FMOCamino acids are available from a variety of sources. The conversion ofthese compounds to the second component pieces of this invention may bereadily achieved by activation of the carboxylic acid group of theN-protected amino acid. Suitable activated carboxylic acid groupsinclude acid halides where X is a halide such as chloride or bromide,acid anhydrides where X is an acyl group such as acetyl, reactive esterssuch as an N-hydroxysuccinimide esters and p-nitrophenyl esters, andother activated intermediates such as the active intermediate formed ina coupling reaction using a carbodiimide such asdicyclohexylcarbodiimide (DCC). Similarly, the corresponding azidoderivative may be prepared by known techniques. In a preferredembodiment, X is hydroxyl for HATU(0-(7-azabenzotriaol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) coupling, or is fluorine for silicon mediatedcoupling.

C. Representative Third Component Pieces

A representative third component piece of this invention is anα,β-unsaturated carboxylic acid or derivative thereof having thefollowing structure 3:

where R₂ and R₅ are as defined above, and P is a carboxylic acidprotective group such as a methyl or t-butyl group. Such third componentpieces may be obtained commercially, or synthesized from thecommercially available aldehyde and the appropriate phosphorusylideaccording to the following reaction scheme:

(see, Wadsworth and Emmons, Org. Syn. 45:44, 1965).

D. Representative Fourth Component Pieces

A representative fourth component piece of this invention is a primaryamine having the following structure 4:

R₁—NH₂   4

where R₁ is as defined above. Suitable fourth component pieces arecommercially available from a variety of sources. Alternatively, thefourth component pieces may be readily prepared by standard organicsynthetic techniques commonly utilized for the synthesis of primaryamines.

Example 2 Combined First-Second Intermediates: The Coupling of First andSecond Component Pieces

The coupling of the component pieces to produce the reverse-turnmimetics of the present invention generally involve the formation ofamide bonds. The amide bonds which link the pieces may be formed bystandard synthetic peptide techniques and may be performed by eitherliquid or solid phase synthesis.

The coupling of the first and second component pieces provides, afterdeprotection, a combined first-second intermediate having the followingstructure 1-2:

where R, R₃, and R₄ are as described above (in this example, R″ ofstructure (I′) is/are hydrogen).

The preparation of a combined first-second intermediate is accomplishedby amide bond formation between the amine of a first component piece 1and the activated carboxylic acid group of a second component piece 2followed by N-deprotection. The synthesis of a representative combinedfirst-second intermediate is depicted schematically below.

In the procedure, to 650 mg (3.17 mmol) first component piece 1aprepared as described in Example 1A and 1 g (3.17 mmol) FMOC-glycinechloride, 2a, 10 ml freshly distilled benzene in a 25 ml argon chargedround bottom flask was added 937 mg (7 mmol) silver cyanide (AgCN), andthe resulting reaction mixture was stirred vigorously for 48 hrs. Thereaction was diluted to 25 ml w/ethyl acetate and filtered through aCelite plug. Volatiles were removed under reduced pressure and theresidue was chromatographed using 20:80 ethyl acetate:hexane as themobile phase over flash grade silica gel to yield 1.1 g (71%) of anamorphous solid.

To 400 mg (0.82 mmol) of the amorphous solid in 5 ml acetonitrile wasadded 1 ml diethylamine (DEA) dropwise and the resulting reactionmixture was stirred at room temperature for 2 hrs. The volatiles wereremoved under reduced pressure and the residue was chromatographed using5% methanol saturated with ammonia 95% dichloromethane as the mobilephase over flash grade silica gel to yield 207 mg (95%) of a combinedfirst-second intermediate, 1-2a, as a thick colorless oil.

Example 3 Combined Third-Fourth Intermediates: The Coupling of Third andFourth Component Pieces

The coupling of a third component piece with a fourth component pieceprovides a combined third-fourth intermediate. The combined third-fourthcomponent piece is produced by amine bond formation resulting from theconjugate addition of the amine group of a fourth component piece 4 tothe α,β-unsaturated carbonyl group of a third component piece 3.

The coupling of third and fourth component pieces provides, afterdeprotection, a combined third-fourth intermediate having the followingstructure 3-4:

where R₁, R₂, and R₅ are as described above (in this example, n ofstructure (I′) is O).

The preparation of a combined third-fourth intermediate is accomplishedby amine bond formation between the primary amino group of a fourthcomponent piece 4 and α,β-unsaturated carbonyl group of a thirdcomponent piece 3 followed by O-deprotection. The synthesis of arepresentative combined third-fourth intermediate is depictedschematically below.

In the procedure, to 5 g of tyramine suspended in 40 ml freshlydistilled tetrahydrofuran (THF) in an argon charged, 250 ml round-bottomflask was added methanol sufficient to dissolve the suspension. To theresulting solution was added 5.3 ml (4.67 g, 36.4 mmol) oft-butylacrylate dropwise over the course of 5 min, and the resultingreaction mixture was stirred overnight at room temperature. Anadditional 2 ml of t-butylactylate was added to consume the remainingstarting material and the reaction was stirred an additional 4 hrs.Volatiles were removed under reduced pressure and the residue waschromatographed using 95:5 dichloromethane:ammonia saturatedmethanol:NH₃/MeOH as the mobile phase over flash grade silica gel toyield 6.6 g (68%) of the ester, a colorless oil which solidified uponovernight refrigeration. To a solution of 1 gram (3.77 mmol) of theester in 20 ml dichloromethane at 0° C. was added 80 ml of coldtrifluoroacetic acid (TFA) and the resulting reaction mixture wasstirred with warming to room temperature over the course of 24 hrs.Volatiles were removed under reduced pressure to yield 950 mg of a clearoil. The end product was dissolved in 95:5 dichloromethane:methanol andslowly filtered through a pad of neutral alumina. Volatiles were removedfrom the filtrate to yield 750 mg of 3-4a as an amorphous solid.

Example 4 Combined First-Second-Third-Fourth Intermediates: The Couplingof Combined First-Second and Third-Fourth Intermediates

The coupling of a combined first-second intermediate with a combinedthird-fourth intermediate provides a combined first-second-third-fourthintermediate. The combined first-second-third-fourth intermediate isproduced by amide bond formation resulting from the coupling of theamine group of a combined first-second intermediate 1-2 to thecarboxylic acid group of a combined third-fourth intermediate 3-4. Thecombined first-second-third-fourth intermediate has the followingstructure 1-2-3-4:

where R, R₁, R₂, R₃, R₄ and R₅ are as described above.

The synthesis of a representative combined first-second-third-fourthintermediate is depicted schematically below.

In the procedure, 212 mg (1.0 mmol) 3-4a, 270 mg (1.01 mmol) 1-2a, and136 mg (1.01 mmol) 1-hydroxybenzotriazole hydrate (HOBT) were dissolvedin 10 ml dimethylformamide (DMF) and cooled to 0° C. To this solutionwas added 290 mg (1.52 mmol, 1.5 eq)1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and the resultingreaction mixture was stirred and warmed to room temperature over thecourse of 24 hours. The DMF was removed under reduced pressure and theresidue was redissolved in 200 ml ethyl acetate. The ethyl acetate layerwas washed with saturated aqueous sodium bicarbonate, water, and driedover anhydrous sodium sulfate. Volatiles were removed under reducedpressure and the residue was chromatographed using 95:5dichloromethane:ammonia saturated methanol as eluent over flash-gradesilica gel to yield 310 mg (0.68 mm 67%) 1-2-3-4a as a thick colorlessoil.

Example 5 The Synthesis of a Representative Reverse-Turn Mimetic:Cyclization of a Combined First-Second-Third-Fourth Intermediate

The cyclization of a combined first-second-third-fourth intermediateprovides a reverse-turn mimetic of the present invention. The combinedfirst-second-third-fourth intermediate 1-2-3-4 is cyclized by treatmentwith camphorsulfonic acid (CSA) or, in a preferred embodiment, TMSOTF(at 0° C.) to provide a reverse-turn mimetic having the followingstructure (Ia):

where R₁, R₂, R₃, R₄, and R₅ are as described above.

The synthesis of a representative reverse-turn mimetic of the presentinvention is depicted schematically below.

In the procedure, 0.5 g (2.4 mmol) camphorsulfonic acid (CSA) wasazeotroped with 3-15 ml portions of freshly distilled toluene and driedunder vacuum at 40° C. for 3 hrs in a 100 ml round-bottom flask equippedwith a reflux condenser. Then 20 ml of freshly distilled toluene wasadded and the CSA solution was heated to a vigorous reflux. To thisrefluxing CSA solution was added a solution of 50 mg (0.11 mmol)1-2-3-4a in 20 ml of freshly distilled toluene by syringe pump over thecourse of 1 hr. The resulting reaction mixture was refluxed for 12 hrs,cooled to room temperature and diluted to 200 ml ethylacetate. Theorganic layer was washed with 2-75 ml portions of saturated aqueoussodium bicarbonate, 75 ml saturated aqueous sodium chloride, and driedover anhydrous sodium sulfate. Volatiles were removed under reducedpressure to yield 22 mg of Ia as a glassine solid. The crude product wastriturated with 50/50 diisopropyl ether:hexane to remove non-polarimpurities. The solid was then dissolved in dichloromethane and filteredto remove polar impurities. The residue upon evaporation was dried invacuo for 24 hrs.

Example 6 Synthesis of a Representative Reverse-Turn Mimetic Salt

The reverse-turn mimetics of the present invention are nitrogen basesand may, therefore, be converted to their corresponding salts bytreatment with various acids. In this example, the preparation of arepresentative salt of a reverse-turn mimetic is described.

The 2,4-dinitrobenzoic acid salt of reverse-turn mimetic Ia, prepared asdescribed in Example 5, was obtained by treatment of the reverse-turnmimetic with the acid in aqueous methanol. In the procedure, 5 mg (12.7μmol) Ia was dissolved in 3 ml of 80/20 methanol:water and cooled to 0°C. To this solution was added 2.70 mg (12.7 μmol, 1.0 eq) 2.4dinitrobenzoic acid, and the resulting solution stirred until it becamehomogenous. Volatiles were removed under reduced pressure and theresidue was dried in vacuo for 24 hrs. The residue was taken up in warmwater and filtered to remove insoluble impurities. The solution was thenlyophilized to give the salt, 5.

Example 7 Synthesis of a Representative Reverse-Turn Mimetics

This example illustrates the synthesis of further representativereverse-turn mimetics of this invention.

Synthesis of structure (6):

To a stirred solution of N-benzylglycine ethyl ester (1.93 g, 10 mmol)in THF (50 mL) was added Boc-Ala-OH (1.9 g, 10 mmol), followed by HOBt(1.62 g, 12 mmol) and EDCI (2.3 g, 12 mmol) at room temperature (“rt”).The resulting solution was stirred at rt for 5 hours (“h”). Afterdilution with EtOAc (100 mL), the solution was washed with 1N HCl (50mL), sat. NaHCO₃ (50 mL), and brine (50 mL); it was dried (MgSO₄),passed through a short pad of SiO₂, and concentrated to give an oil inquantitative yield. TLC showed that the product was pure enough for usein the next reaction without further purification. TLC R_(f) 0.6(hexane:EtOAc=5:5); ¹H NMR (CDCl₃) {the spectrum was assigned as 2:1mixture of rotamers} δ 1.24 (two t, 3H, J=6.5 Hz), 1.35 and 1.36 (two d,3H, J=6.5 Hz), 1.42 and 1.43 (two s, 9H), 3.80 (dd, 1H, J=18 Hz), 4.15(q, 2H, J=6.5 Hz), 4.40 (dd, 1H), 4.65 (ABq, 2H, J=16.5 Hz), 4.80 (m,1H), 5.40 (two d, 1H, J=8 Hz, NH), 7.1-7.3 (m, 5H, phenyl); MS ES+365.1(M+H⁺).

Synthesis of structure (7):

To a stirred solution of 3.8 g of crude ethyl ester (6) in THF/H₂O(50/50 mL) was added LiOH H₂O (1 g) at rt. After 30 min stirring at rt,the solution was washed with Et₂O (50 mL) and aqueous phase wasacidified by 6N HCl (pH 2), and extracted with EtOAc (3×100 mL). Thecombined organic extracts were dried (MgSO₄), passed through a short padof SiO₂, and concentrated to provide a foam in quantitative yield. Theproduct was used for the next reaction without further purification. ¹HNMR (CDCl₃) {mixture of rotamers} δ 1.33 (two d, 3H, J=7 Hz), 1.41 (twos, 9H), 3.8-4.8 (set of m, 5H), 5.70 (two d, 1H, J=8 Hz, NH), 7.2-7.6(m, 5H, phenyl).

Synthesis of structure (8):

To a stirred solution of 3.4 g of acid (7) and cyanomethylenetriphenylphosphorane (4.1 g, 12 mmol) in dichloromethane (100 mL) wasadded sequentially DIEA (5 mL, 30 mmol), DMAP (250 mg, 2 mmol), and EDCI(2.9 g, 15 mmol) at rt. After 12 h stirring, the solution wasconcentrated, and the resulting residue was taken up in 1N HCl (100 mL)and extracted with EtOAc (3×100 mL). The combined extracts were washedwith sat. NaHCO₃ (100 mL), dried (MgSO₄), passed through a short pad ofSiO₂, and concentrated. The crude product was purified by flashchromatography (hexane:EtOAc=50:50 to 30:70 to 20:80) to provide a foamysolid (4.40 g, 71%). TLC R_(f) 0.5 (EtOAc); ¹H NMR (CDCl₃) {mixture ofrotamers} δ 1.28 (two d, 3H, J=6.5 Hz), 1.44 (two s, 9H), 4.2-4.7 (setof m, 5H), 5.5 (two d, 1H, J=8 Hz, NH), 7.2 (m, 5H), 7.5-7.8 (m, 15H);MS ES+m/z 520.3, 620.3 (M+H⁺).

Synthesis of structure (9):

To a stirred solution of the phosphorane (8) (310 mg, 0.5 mmol) indichloromethane (5 mL) was bubbled O₃ at −78° C. for 15 min untilsolution became greenish blue; TLC showed complete consumption of thestarting material. After bubbling Ar to remove excess ozone from thissolution, N-benzylglycine ethyl ester (100 mL) was added, and thesolution was stirred at −78° C. for 30 min. After concentration, theresidue was dissolved in EtOAc (50 mL), washed with 1N HCl (20 mL), sat.NaHCO₃ (20 mL), brine (20 mL), dried (MgSO₄), and concentrated again.The crude product was purified by flash chromatography(hexane:EtOAc=90:10 to 80:20 to 70:30 to 60:40) to provide an oil (105mg, 39%). TLC R_(f) 0.42 (hexane:EtOAc=60:40); ¹H NMR (CDCl₃) {thespectrum was assigned as a 1:1 mixture of rotamers} δ 1.25 (two t, 3H,J=7 Hz), 1.31 and 1.38 (two d, 3H, J=7 Hz), 1.41 and 1.43 (two s, 9H),3.8-4.8 (set of m, 11H), 5.5 (two d, 1H, NH), 7.2-7.4 (m, 5H). MS ES+m/z440.3, 540.3 (M+H+).

Synthesis of structure (10):

A solution of 100 mg ketoamide (9) (0.18 mmol) in 0.5 mL dichloromethanewas treated with 0.5 mL TFA at rt for 30 min. After concentration, theresidue was dissolved in MeOH (2 mL) and treated with ZnCl₂ (6 mg) andNaBH₃CN (15 mg) at rt for overnight 13 h). After concentration, theresidue was taken up in sat. NaHCO₃ (20 mL), extracted with EtOAc (2×20mL). The combined organic extracts were dnred (MgSO₄), concentrated toan oil, and purified by preparative TLC (hexane:EtOAc=60:40) to providea glassy solid (52 mg, 77%). (The enamine proved resistant to reductionby this method.) TLC R_(f) 0.58 (EtOAc); ¹H NMR (CDCl₃) δ 1.41 (d, 3H,J=6.5 Hz, CHCH₃), 3.93 (ABq, 2H, J=18 Hz, CH2 in Gly), 4.46 and 4.75(ABq, 1H each, J=14.5 Hz, CH₂Ph), 4.76 (ABq, 2H, J=14 Hz, CH₂Ph), 5.22(q, 1H, J=7 Hz, CHCH₃), 6.83 (s, 1H, ═CH), 7.33 (m, 10H, phenyls); ¹³CNMR (CDCl₃) δ 16.63, 49.59, 49.66, 49.84, 50.98, 111.92, 119.16, 128.07,128.22, 128.29, 128.52, 128.94, 128.97, 134.78, 134.43, 157.96, 160.67,165.33. MS ES+m/z 376.3 (M+H⁺).

Synthesis of structure (11):

A solution of 25 mg structure (10) (0.066 mmol) with PtO₂ (5 mg) in MeOH(2 mL) was stirred under H₂ atmosphere (20 atm) for 10 days. Afterconcentration, the residue was purified by preparative TLC(hexane:EtOAc=60:40 to 50:50) to yield a pale yellow oil (14 mg, 56%)with starting material (10 mg). TLC Rf 0.49 (EtOAc); ¹H NMR (CDCl₃) δ1.14 (d, 1.5H, J=7 Hz, CHCH₃), 1.52 (d, 1.5H, J=7 Hz, CHCH₃), 3.2-4.8(set of m, 10H), 7.33 (m, 10H, phenyls); MS ES+m/z 378 (M+H⁺). RP-HPLCanalysis: C-18; A: 0.1% TFA (aq); B 0.1% TFA (CH₃CN); gradient:0-90%/40′; 254 nm tR 24.1′ and 24.7′ showed a 2:1 ratio.

Example 8 Synthesis of a Representative Reverse-Turn Mimetics

This example further illustrates the syntheses of reverse-turn mimeticsof this invention. Specifically, the preparation of [4.4.0] bicyclicreverse-turn mimetics was carried out in solution phase (Method A) andon solid phase (Methods B and C). Structures of representative mimeticsare given in Table 2. The solid phase syntheses of these reverse-turnmimetics demonstrate that libraries containing such members may bereadily prepared.

Method A solution phase synthesis is analogous to the solid phasesynthesis of Method B and was carried out essentially as illustrated inFIG. 2. The compounds were purified as in Method C, below.

The solid phase synthesis of Method B is illustrated in FIG. 8.Referring to that figure, commercially available aminomethyl resin wasreacted with excess 4-bromo-2-butenoic acid and DIC(diisopropylcarbdiimide) in DMF to give 4-bromo-2-butenamide resin.Substitution of the bromo group with a primary amine in DMSO gave thecorresponding 4-alkylamino-2-butenamide resin. Standard peptide couplingprocedures on solid phase were performed to giveN-alkyloxycarbonyl-α-alkyl-β-alanyl-α-alkylglycyl-N′-alkylamino-2-butenamideresin. The reverse-turn mimetics were obtained by osmium tetroxidecatalyzed periodate oxidation of the resin followed by the treatment ofthe resulting monocyclic product with a catalytic amount of TFA indichloromethane. The crude products gave a single major peak byreverse-phase HPLC analysis.

The solid phase sythesis of Method C is similar to Method B and is givenin Example 11 and illustrated in FIG. 9. Selected compounds werepurified by flash chromatography or preparative TLC on silica gel usingsuitable combinations of EtOAc and MeOH.

The mimetics were characterized as follows: Analytical C₁₈ reverse-phaseHPLC was carried out using standard techniques (mobile phase: gradientsof 0.1% in water and acetonitrile. By these methods, crude productssynthesized on solid phase generally displayed purities of greater than80%, and all purified compounds greater than 95%. Electrospray massspectrometry was carried out using standard techniques. The observedvalue of the (M+H⁺) ion is given for each compound in Table 2. ¹H NMRwas carried out on purified mimetics and spectra were assigned by acombination of COSY and ROESY experiments. All spectra were consistentwith the structures indicated below, and displayed a conformationsimilar to a type I or type II β-turn.

TABLE 2 Representative Reverse-Turn Mimetics

MS No. R R₂ R₃ R₄ Method (MH⁺) 12 Bn H Me Me A, B 332 13 p-MeO—Ph(CH₂)₂H H Bn A 438 14 p-MeO—Ph(CH₂)₂ H H Phenethyl A 452 15 p-OH—Ph(CH₂)₂ H HPhenethyl A 438 16 p-OH—Ph(CH₂)₂ H Bn Pentyl A 494 17 i-Bu H (CH₂)₂CO₂HiBu A 398 18 i-Bu H CH₂CO₂H iBu A 384 19 i-Bn Bn Bn Pentyl A 554 20 Bn HMe Bn B 408 21 Bn H Bn Bn B 484 22 Bn H Me n-Bu B 374 23 Bn H Bn n-Bu B449 24 Bn H Me i-Amyl B 388 25 Bn H Bn i-Amyl B 469 26 Bn H Bn p-Cl—Bn C518 27 Bn Ac—NH Me Me C 389 27 Bn Bz—NH Me Me C 451 29 p-OH—Ph(CH₂)₂ HBn Phenethyl C 515 30 p-OH—Ph(CH₂)₂ H Phenethyl Pentyl C 508 31p-OH—Ph(CH₂)₂ H Bn 2-Pyr(CH₂)₂ C 529 32 Bn H t-BuO₂C—(CH₂)₂ Me B 446 33*Ph H Bn c.HexCH₂ C 496 34* Ph Ac—NH Me Me C 395 35* p-Tolyl H Bn p-Cl-BnC 538 36 p-OH—Ph(CH₂)₂ H H Pentyl C 404 37 p-OH—Ph(CH₂)₂ H Me Pentyl C418 38 p-OH—Ph(CH₂)₂ H MeS(CH₂)₂ Pentyl C 478 39 p-OH—Ph(CH₂)₂ H i-BuPentyl C 460 40 p-OH—Ph(CH₂)₂ H i-Pr Pentyl C 446 41 p-OH—Ph(CH₂)₂ Hs-Bu Pentyl C 460 42 p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ Pentyl C 510 43p-OH—Ph(CH₂)₂ H Ph Pentyl C 480 44 p-OH—Ph(CH₂)₂ H p-Cl—PhCH₂ Pentyl C528 45 p-OH—Ph(CH₂)₂ H p-NH₂—PhCH₂ Pentyl C 509 46 p-OH—Ph(CH₂)₂ H BnMeO(CH₂)₃ C 496 47 p-OH—Ph(CH₂)₂ H Bn i-Amyl C 494 48 p-OH—Ph(CH₂)₂ H BnHeptyl C 522 49 p-OH—Ph(CH₂)₂ H Bn Bn C 514 50 p-OH—Ph(CH₂)₂ H Bn c.HexCH₂ C 520 51 p-OH—Ph(CH₂)₂ H Bn 4-PyrCH₂ C 515 52 p-OH—Ph(CH₂)₂ H Bni-Bu C 480 53 p-OH—Ph(CH₂)₂ H Bn 3,4-MeO- C 588 Phenethyl 54p-OH—Ph(CH₂)₂ H Bn N-Pyridone- C 549 (CH₂)₃ 55 p-OH—Ph(CH₂)₂ Hp-OH—PhCH₂ MeO(CH₂)₃ C 512 56 p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ i-Amyl C 510 57p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ Heptyl C 538 58 p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ BnC 530 59 p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ c.Hex CH₂ C 536 60 p-OH—Ph(CH₂)₂ Hp-OH—PhCH₂ 4-PyrCH₂ C 531 61 p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ i-Bu C 496 62p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ 3,4-MeO- C 604 Phenethyl 63 p-OH—Ph(CH₂)₂ Hp-OH—PhCH₂ N-Pyridone- C 565 (CH₂)₃ 64 p-OH—Ph(CH₂)₂ H p-OH—PhCH₂Phenethyl C 544 66 p-OH—Ph(CH₂)₂ H Phenethyl Phenethyl C 529 67p-OH—Ph(CH₂)₂ H p-OH—PhCH₂ 2-Pyr(CH₂)₂ C 545 68 p-OH—Ph(CH₂)₂ HPhenethyl 2-Pyr(CH₂)₂ C 543 69 p-OH—Ph(CH₂)₂ H i-Pr i.Amyl C 446 70p-OH—Ph(CH₂)₂ H i-Bu i.Amyl C 460 *—SO₂— replaces —OC(O)— in the R₁ sidechain in this compound.

Example 9 Activity of a Representative Reverse-Turn Mimetic in OpioidReceptor Binding

In this example, the binding activity of representative reverse-turnmimetics to the delta (δ) and mu (μ) opioid receptors as well as to apreparation of non-selective opioid receptors is described. The bindingaffinity of 5, the 2,4-dinitrobenzoic acid salt of reverse-turn mimeticof structure Ia (prepared as described in Example 6), and a variety ofreverse-turn mimetics prepared as described in Example 8, was evaluatedin these competitive radioligand binding assays.

A. Opiate (δ) Binding Activity

In this method, membranes were prepared from whole brains of male guineapigs and equilibrated with 2 nM [³H]DPDPE (D-pen³, D-pen⁵) enkephalinfor 1 hour at 4° C. after which test substances were added and incubatedfor 4 hours at 25° C. Non-specific binding was determined in thepresence of 0.3 μM naltrindole. Bound [³H]DPDPE was separated from freeradioligand by rapid filtration through glass fiber filtermats andsubsequently washed 3. times. Filtermats were then counted in the LKBBetaplate to determine specifically bound [³H]DPDPE. (See Mosberg etal., “Structural Requirements for δ Opiate Receptor Binding,” Molec.Pharmacol. 31:599-602, 1987.)

TABLE 3 Effect of Reference Compounds on [³H]DPDPE Bound (2nM) CompoundIC₅₀(nM) Ki (nM) Hill Coefficient DAMGO 4,800 1,200 1.08 DPDPE 5.5 1.30.86 Naltrindole 0.63 0.20 0.53 U-50488 53,000 16,000 0.73

In this assay, the radioligand, [³H]DPDPE, was determined to have aK_(d)=0.65 nM with a B_(max)=12.6 fmol/mg protein and a specific bindingof 60%. At a concentration of 10 μM, 5 was found to inhibit radioligandbinding at the 60% level, and exhibited a K_(i)=1.7±0.3 μM and anIC₅₀=6.9±1.2 μM. These results are presented in FIG. 1 (o) which depictsthe % inhibition of radioligand binding as a function of reverse-turnmimetic 5 concentration. Also, at a concentration of 10 μM, reverse-turnmimetic 16 was found to inhibit radioligand binding at the 92% level.These results demonstrate that reverse-turn mimetics 5 and 16, inparticular, and the reverse-turn mimetics of the present invention, ingeneral, effectively inhibit binding to the δ opiate receptor, andpossess analgesic activity.

B. Opiate (μ) Binding Activity

In this method, membranes were prepared from whole brains of male guineapigs and incubated with 2 nM [³H]DAMGO (D-Ala², N-methyl-phe⁴,gly-ol⁵)-enkephalin) for 2 hours at 25° C. Non-specific binding wasdetermined in the presence of 0.5 μM DAMGO. Bound [³H]DAMGO wasseparated from free radioligand by rapid filtration through glass fiberfiltermats and subsequently washed 3 times. Filtermnats were thencounted in the LKB Betaplate to determine specifically bound [³H]DAMGO.(See Patricia et al., “Pharmacological profiles of fentanyl analogs atμ, δ and κ opiate receptors,” Eur. J. Pharmacol. 213:219-225, 1992.)

TABLE 4 Effect of Reference Compounds on [³H]DAMGO Bound (2nM) CompoundIC₅₀(nM) Ki (nM) Hill Coefficient DAMGO 6.5 0.59 0.92 DPDPE 4.0 0.371.32 Fentanyl 14 1.2 0.99 Naloxone 9.3 0.76 1.09 Naltrindole 27 2.5 0.98Norbinaltorphimine 280 26 1.13 U-50488 6.1 0.59 0.70

In this assay, the radioligand, [³H]DAMGO, was determined to have aK_(d)=0.27 nM with a B_(max)=8.7 pmol/mg protein and a specific bindingof 70%. At a concentration of 10 μM, 5 inhibited radioligand binding atthe 64% level, and exhibited a K_(i)=0.64±0.08 μM and an IC₅₀=54±0.7 μM.These results are presented in FIG. 1 () which depicts the % inhibitionof radioligand binding as a function of reverse-turn mimetic 5concentration. Also, at a concentration of 10 μM, reverse-turn mimetic16 was found to inhibit radioligand binding at the 98% level. Theseresults demonstrate that reverse-turn mimetics 5 and 16, in particular,and the reverse-turn mimetics of the present invention, in general,effectively inhibit binding to the μ opiate receptor, and possessanalgesic activity.

C. Opiate (Non-selective) Binding Activity

In this method (Childers et al. Eur. J. Pharmacol. 55: 11, 1979),membranes were prepared from rat cerebral cortex and incubated with[³H]naloxone (1 nM) and β-turn mimetics (30 μM-0.3 nM) for 40 min at 22°C. Following incubation, the membranes were rapidly filtered undervacuum through glass fiber filters (Filtermat A,. Wallac). The filterswere then washed several times with an ice-cold buffer using a cellharvester (Tomtec). Bound radioactivity was measured with ascintillation counter (Betaplate, Wallac) using solid scintillant(MultiLex B/HS, Wallac). In same experiment, the reference compound(naloxone) was tested at eight concentrations in duplicate to obtain acompetition curve in order to validate this experiment.

The specific radioligand binding to the receptors is defined as thedifference between total binding and nonspecific binding determined inthe presence of an excess of unlabelled ligand. Results were expressedas a percent of control specific binding obtained in the presence ofβ-turn mimetics. IC₅₀ values and Hill coefficients (nH) were determinedby non-linear regression analysis of the competition curves. Theseparameters were obtained by Hill equation curve fitting. The inhibitionconstants (Ki) were calculated from the Chen Prusoff equation(Ki=IC₅₀/(1+L/K_(d)), where, L=concentration of radioligand in theassay, and K_(d)=affinity of the radioligand for the receptor).

In this assay, the radioligand, [³H]naloxone, was determined to have anIC₅₀=2.5 nM. Reverse turn mimetics, prepared and purified as describedin Example 8, displayed specific binding of up to 99% at 1 μMconcentration. Compounds 29 and 30 were determined to have IC₅₀s of 80and 27 nM, respectively, with Hill coefficients of 0.9. These resultsdemonstrate the mimetics 29 and 30, in particular, and the reverse-turnmimetics of the present invention, in general, effectively inhibitbinding to the opioid receptor (non-selective), and possess analgesicactivity.

Example 10 In Vivo Activity of a Representative Reverse-Turn Mimetic forAnalgesic Activity

In this example, the in vivo activity of a representative reverse-turnmimetic as an analgesic agent is presented. Compound 5, prepared asdescribed in Example 6 (hereinafter referred to as “test compound”), wasutilized in the mouse tail flick assay (PanLabs, Pharmascreen Test No.10402A). In this assay, the time required to elicit a tail-flickresponse to radiant heat pain stimulus in a group of mice is measured asthe pain threshold response.

Groups of five (3 test groups+1 saline control+1 morphine positivecontrol) male ICR mice weighing 22 (±2) grams each were used. Each ofthese animals were pre-selected and elicited a tail flick responsewithin 6-7.5 seconds after a focused beam of radiant heat was focused onthe middle dorsal surface of the animal's tail. Specific amounts of thetest compound (i.e., 10, 30 and 100 μg) were dissolved in 5 microliters(5 μl) saline containing 6% DMSA and administeredintracerebroventricularly (ICV) to each animal. A saline-only solutionwas used as a negative control, with an ICV injection of 10 μg/5μl/animal of morphine serving as a positive control.

At one minute post-ICV injection, the groups of mice were measured fortail flick response, with a maximum cut-off time of 15 seconds. The meanof the response time for each treatment groups was calculated for acomparison between pre-treatment (“0 time”) and 1 minute post-treatment1 (“1 min.”). Prolongation 1 minute post-treatment of over 50% (“%Prolong.”) was considered significant activity. The results of thisexperiment are presented in Table 5, and demonstrate that the testcompound had significant analgesic activity (i.e., approximately 10%-15%the potency of morphine).

TABLE 5 In Vivo Tail Flick Assay Compound Dose/5 μl 0 Time 1 Min. %Prolong. Saline  0 6.9 6.7 — 6.9 7.5 — 6.1 6.2 — 6.5 6.3 — Avg = 6.6Avg. = 6.7  2% Morphine  10 μg 7.5 >15 — 6.3 >15 — 7.2 >15 — 6.8 >15 —Avg. = 7.0 Avg. > 15 100% Test Compound 100 μmg 6.5 >15 — 6.3 >15 —6.5 >15 — 6.8 >15 — Avg. = 6.5 Avg. > 15 100%  30 μg 6.5 >15 — 6.7 7.2 —7.2 6.3 — 6.3 >15 — Avg. = 6.7 Avg. > 15  63%  10 μg 6.5 7.5 — 7.2 7.5 —6.9 6.7 — 6.2 6.8 — Avg. = 6.7 Avg. 7.1  6%

Example 11 Synthesis of Representative Reverse-Turn Mimetics

This example further illustrates the synthesis of reverse-turn mimeticsof this invention. Specifically, the preparation of [4.4.0] bicyclicreverse-turn mimetics was carried out on solid phase by a methodalternative to that of Example 8, method B. The method is outlined inFIG. 9.

Synthesis of 2-Bromo-1-ethoxy-ethyl-1-oxy-linked Resin (27)

In general, a batch of resin (ArgogelOH or hydroxymethyl polystyrene)was refluxed in 1,2-dichloroethane (DCE) for 4 hours in the presence of8 equivalents of bromoalkylaldehyde diethyl acetal and 2 equivalents ofpyridinium p-toluenesulfonate (PPTS). In one instance, hydroxymethylpolystyrene (10.0 g, 0.7 mmol OH/g, 7 mmol) and 3.5 g of PPTS (14 mmol)were suspended in 200 ml of DCE. Then, a solution of 8.5 ml of2-bromodiethoxyethane (ca. 56 mmol) in DCE (100 ml) was added withstirring and the reaction mixture was heated at reflux (approx. 80° C.).After 4 hours the resin was filtered off and washed with 100 mLdimethylformamide (DMF), 50 mL dimethylsulfoxide (DMSO), 100 mL DMF, 200mL dichloromethane (DCM), 50 mL 1,4-dioxane and finally with 100 mLmethanol. After drying, 11.73 g, of resin 27 was obtained. Bromineanalysis indicated quantitative loading.

Synthesis of Representative Compounds of Structure (Ia′)

Reactions were carried out in plastic disposable syringes of theappropriate size, each fitted with a polypropylene frit to retain theresin. After each step, resin batches were washed with DMF (3×) and DCM(3×). Typically, a 0.03 mmol sample of resin 27 (e.g., 50 mg ofpolystyrene resin with loading of 0.6 mmol Br/g), pre-swollen in DMF,was treated with 1 mL of a 2.0 M solution of amine R₄—NH₂ (2 mmol) inDMSO at 60° C. for 16-24 hrs.

Next, the resin was reacted with 0.09 mmol of Fmoc amino acid(FmocNH—CHR₃—COOH) in the presence of HATU (34 mg, 0.09 mmol) and DIEA(0.032 ml, 0.18 mmol) in DMF (1 mL) until the chloranil test wasnegative (typically 1-2 h). Subsequently, the Fmoc protection wasremoved by treatment with a 25% (v/v) piperidine/DMF solution (2 mL)over 20 min.

The resin was then reacted with 0.09 mmol of an Fmoc beta-amino acid(FmocNH—CHR₅—CHR₂—COOH) in the presence of DIC (0.014 ml, 0.09 mmol) andHOBt (14 mg, 0.09 mmol) in DMF (1 mL) until the Kaiser test was negative(typically 1 hour). The resin was again treated with 25% (v/v)piperidine/DMF solution (2 mL) over 20 min.

Finally, the resin-bound sequence was terminated by reaction withsulfonyl chloride (R₁SO₂Cl, 0.3 mmol) in the presence of DIEA (0.106 mL,0.6 mmol) in DCM (1 mL) for 1 hr (Kaiser test negative). Alternatively,chloroformate R₁OCOCl or isocyanate R₁NCO (the latter does not requirepresence of DIEA) was used instead of sulfonyl chloride for introductionof the R₁ moiety.

The washed and dried resin was re-swollen in DCM, drained and treatedwith 1 mL of formic acid (96%) overnight at rt. In a number of cases, anelevated temperature up to 60° C. or an extended reaction time wasnecessary to complete the cyclization (for conditions see Table 2below). The supernatant was collected and combined with washes (2×0.5 mLof formic acid). The residue obtained after evaporation of formic acidwas redissolved in acetonitrile/water 50:50 mixture, frozen andlyophilized.

Table 6 presents representative compounds of this invention synthesizedby the above procedure.

TABLE 6 Representative Reverse-Turn Mimetics

No. R₁ R₂ R₃ 28

29

30

31

32

33

34

35

36

37

38

39

40

No. R₄ R₅ (M + H⁺) 28

406.3 29

512.3 30

602.4 31

496.3 32

600.3 33

584.3 34

536.4 35

583.6 36

597.6 37

569.4 38

583.4 39

550.5 40

495.3

Example 12 Activity of Representative Reverse-Turn Mimetics in a CellAdhesion Assay

An assay measuring the ability of the compounds of Example 1 toantagonize binding of CS1 peptide to α₄β₁ integrin was performed. Amodification of the procedure of Vanderslice, P. et al. (J. Immunol.,1997, 1710-1718) (incorporated herein by reference) was utilized.

In brief, 100 μL/well of a solution of biotinylated CS1 peptide (1mg/100 mL of phosphate buffered saline (PBS)) was incubated in aNeutrAvidin plate (Pierce) for 1 h at room temperature. The plate wasthen washed 3× with distilled water and treated with 200 μL of blockingbuffer (3% BSA in PBS) for at least 4 h. Blocked plates were washed asabove. Harvested Ramos cells (10⁷/mL) were resuspended in PBS containing10 μL of calcein AM/mL and incubated 30 min in the dark. This suspensionwas diluted with 45 mL PBS and the cells harvested by centrifugation andaspiration. The cells were resuspended in binding buffer (˜5×10⁵/mL). Ifcell lysis was to be monitored ethidium homodimer was added to thebuffer to a final concentration of 5 μM. A solution (10 μL) of compoundto be tested or control peptide was added to appropriate wells followedby 90 μL of the cell suspension. The plate was incubated at 37° C. for 1h. When ethidium homodimer was added, fluorescence at 535/617 wasmeasured before rinsing. Otherwise, the plate was washed 3×, 50 μL oflysis buffer was added to each well, the plate rocked in the dark for 10min, and the fluorescence monitored at 485 nm excitation and 535 nmemission.

Compounds prepared in Example 11 displayed activity in this assay. Assuch, the compounds of this invention effectively inhibit cell adhesionand possess activity as anti-inflammatory agents.

It will be appreciated that, although specific embodiments of theinvention have been described herein for the purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptby the appended claims.

What is claimed is:
 1. A compound having the structure:

wherein Y is selected from -A-N(R₁)—CH(R′)—, -A-N(R₁)—C(═O)—,-A-C(═O)—N(R₁)—, -A-CH(R₁)—O— and -A-CH(R₁)—N(R′)—; A is —(CHR′)_(n)—,where n=0, 1 or 2; B is —(CHR″)_(m)—, where m=1, 2 or 3; R′, R″, R₂, R₃and R₅ are the same or different and independently selected from anamino acid side chain moiety or derivative thereof, a linker and a solidsupport; and R₁ and R₄ represent the remainder of the compound; andwherein any two adjacent CH groups or adjacent NH and CH groups on thefused bicyclic ring may optionally form a double bond.
 2. The compoundof claim 1 wherein Y is -A-N(R₁)—CH(R′)— and the compound has thestructure:


3. The compound of claim 2 wherein two adjacent CH groups on the fusedbicyclic ring form a double bond and the compound has the structure:


4. The compound of claim 3 wherein A is —(CH₂)_(n)—, B is —(CH₂)_(m)—,R′ is hydrogen and the compound has the structure:


5. The compound of claim 1 wherein Y is -A-N(R₁)—C(═O)— and the compoundhas the structure:


6. The compound of claim 5 wherein A is —(CH₂)_(n)—, B is —(CH₂)_(m)—and the compound has the structure:


7. The compound of claim 1 wherein Y is -A-C(═O)—N(R₁)— and the compoundhas the structure:


8. The compound of claim 7 wherein A is —(CH₂)_(n)—, B is —(CH₂)_(m)—and the compound has the structure:


9. The compound of claim 1 wherein Y is -A-CH(R₁)—O— and the compoundhas the structure:


10. The compound of claim 9 wherein A is —(CH₂)_(n)—, B is —(CH₂)_(m)—compound has the structure:


11. The compound of claim 1 wherein Y is -A-CH(R₁)—N(R′)— and thecompound has the structure:


12. The compound of claim 11 wherein two adjacent NH and CH groups onthe fused bicyclic ring form a double bond and the compound has thestructure:


13. The compound of claim 12 wherein A is —(CH₂)_(n)—, B is —(CH₂)_(m)—and the compound has the structure:


14. A compound having the structure:

wherein Y is —CH(R₅)-A-N(R₁)—; A is —(CHR′)_(n)—, where n=0, 1 or 2; Bis —(CHR″)_(m)—, where m=1, 2 or 3; R′, R″, R₂, R₃ and R₅ are the sameor different and independently selected from an amino acid side chainmoiety or derivative thereof, a linker and a solid support; and R₁ andR₄ represent the remainder of the compound; and wherein any two adjacentCH groups or adjacent NH and CH groups on the fused bicyclic ring mayoptionally form a double bond.
 15. The compound of claim 14 wherein A is—(CH₂)_(n)—, B is —(CH₂)_(m)— and the compound has the structure:


16. The compound of claim 15 wherein n is 0, m is 1 and the compound hasthe structure:


17. A composition comprising a compound of claim 1 or claim 14 incombination with a pharmaceutically acceptable carrier or diluent.
 18. Alibrary of compounds, comprising at least one compound of claim 1 orclaim
 14. 19. A method of identifying a biologically active compound,comprising contacting the library of claim 18 with a target to detect orscreen the biologically active compound.